利用游離DNA檢測癌症病患體細胞突變

Abstract

目前對於癌症細胞的檢測方式大多具有侵入性、短期內無法連續性使用、化療或手術治療後具有監控上困難的特性，若能以病患接受度較高，及方便取得檢體的非侵入性方式做為檢測方法，不僅可以提高病患接受檢測意願外，也可做為癌症監控的工具。過去研究發現，當細胞凋亡後，會釋放出細胞內的物質到人體體液中，其中一種微量小片段的DNA，稱之為cell free DNA(cfDNA)；由癌細胞釋放出來的DNA稱之為circulating tumor DNA(ctDNA)。液態組織切片(liquid biopsy)就是從周邊血裡得到的游離DNA，相較於血液腫瘤指數提供更準確的檢測數據，尋找癌症上的特異點位可以幫助標靶治療找出更適合的藥物。利用次世代定序技術分析方法(Next Generation Sequence, NGS)，針對血液游離DNA與腫瘤切片組織的關聯性，設計一個組合包含與癌症有相關的50個常見和癌症相關的基因做為篩檢平台，並建立聚合酶連鎖反應及NGS，用以監控癌症突變的變化量。若能在癌症病患的血液游離DNA與腫瘤切片組織中找到相同特定基因突變點位，並且正常人在此突變點位沒有變異的話，就可以使用此篩檢平台，做為連續性常規的癌症監控工具。本研究顯示石蠟包埋切片試驗結果與循環腫瘤DNA之檢驗結果具有相關性，未來也希望應用到像是尿液、糞便及其他體液中，透過萃取液態組織中的ctDNA來檢測相關器官的癌症病患，而可以檢測出癌症基因突變。

The current methods for early detection of a tumor are usually invasive. It remains difficult using the methods to trace the tumor proliferation longitudinally. To overcome these hurdles would render the public acceptance as a biomarker in clinical practice. The growing evidences demonstrated that either the cells or cell components released from the tumors could be detected in the body fluids. The DNA fragments from cells (cfDNA) can be detected from the plasma or serum, like the fetal DNA in the maternal plasma. The DNA from the shedding tumor cells in the plasma is called circulating tumor DNA (ctDNA). The somatic mutations as well as its reads from ctDNA shown in next generation sequencing (NGS) results can be employed as a specific tumor biomarker. More and more studies confirmed that the detection rate from ctDNA is superior to the protein biomarkers. In the study, we validate the feasibility using the NGS to detect the somatic mutation hot spots from ctDNA in five patients with different tumors. In addition to using NGS for detection, we also tested the designed amplicon-based method after the identification of the somatic mutation. While compared to NGS method, the amplicon-based sequencing can also identify the mutations. However, the amplicons can only be designed after the initial screening by NGS. In conclusion, NGS to sequencing the somatic mutation from a gene panel is reliable; however, to expand more cancer-related genes in the panel would make the method with higher detection rate. In the long run, it should be applicable by sampling the ctDNA from other body fluids such as urine, cerebrospinal fluid or even tears.